PROJECT SUMMARY Each year, approximately 8 million children under the age of five die, and approximately 3.3 million of those deaths occur in the neonatal period. Infections are the single largest contributor to these deaths, accounting for an estimated 36% of the mortality. However, our understanding of how the neonatal and infant immune system influence this susceptibility to infectious diseases is limited. Thus, to elucidate mechanisms that regulate the development of infant immunity, we propose evaluating immune development in a setting of high risk of neonatal and infant infection. In particular, infants who are exposed to HIV, yet remain uninfected, suffer increased rates of respiratory and diarrheal illnesses. Feeding status is also associated with altered risk of infection, with exclusively breastfed children less likely to suffer diarrheal illness and death. Our preliminary data suggest that both HIV exposure and feeding status influence the infant microbiome, which is increasingly recognized for its important role in driving immune development. Thus, to better understand the unique characteristics of immune development in neonates and infants, we propose evaluating the impact of HIV exposure and feeding status on the interplay between the microbiome, innate and adaptive cellular immune ontogeny, and vaccination outcomes. Our preliminary data also suggests that HIV exposure leads to oligoclonality in the T cell repertoire, potentially narrowing the spectrum of antigens to which infant T cells can respond. We have also found that HIV infection increases natural killer (NK) cell diversity while decreasing NK cell cytotoxic function. Using longitudinal samples collected from HIV-exposed uninfected (HEU) and HIV- unexposed (HU) babies in an area of extremely high HIV prevalence in South Africa, we will: 1) compare the T and NK cell repertoires and function between HEU and HU babies, 2) determine the impact of maternal HIV, feeding status, gut and breastmilk microbiome on the infant mucosal microbiome, and 3) build a predictive model of effective pertussis and rotavirus vaccines to identify the major factors that associate with vaccine success or failure. This study will provide a comprehensive understanding of how cellular immune responses and the microbiome evolve in the first year of life and influence the ability to generate an effective cellular and humoral immune responses.